POLI-CLINICO
9. PROCESO CONCEPTUAL DEL SISTEMA TÉCNICO CONSTRUCTIVO
2.2.1 Rationale of design for elastin-like polypeptide nanoparticles
The first step in the creation of self-assembled and functionalized ELP-based nanoparticles was the choice or design of suitable sequences. On the one hand, a monomer to micelle transition temperature well below 37 °C had to be chosen to gain stable particles at physiological conditions. This transition temperature had to, on the other hand, be above 4 °C in order to allow disassembly for purification and modification. A micelle to aggregate transition temperature above 37 °C was necessary to avoid aggregation during formulation and application. However, it should be low enough to allow purification by selective precipitation upon addition of electrolytes and/or increase in temperature. Amino acids were chosen that did not contain side chains that react with commonly used moieties such as maleimides, succinimidyl esters or isothiocyanates to allow selective modifications of the N- and C-terminus.
Based on this and other reports of micellar systems composed of ELPs[32–36], we selected isoleucine as the guest residue for the ‘hydrophobic’, core-forming block. A length of 60 pentapeptides was chosen to ensure a transition temperature well below 37 °C down to low micromolar concentrations at experimental ‘physiological’ conditions, id est phosphate buffered saline pH 7.2 – 7.4. The transition temperature was expected to be above 4 °C up to millimolar concentrations, such as during formulation and aqueous modification protocols. As guest residues for the micelle corona or ‘hydrophilic’ block, we chose a ratio of alanine and glycine of 3 to 2 with a length of 60 pentapeptides. This should yield micelles that are stable up to 50 °C, therefore being hydrophilic enough to prevent undesired aggregation without prohibiting purification protocols that rely on the aggregation of micelles. The 1:1 ratio of ELP block lengths was expected to lead to the formation of micelles with radii around 20-40 nm. As in literature the correlation between theoretically predicted and actually measured aggregation numbers was not very straightforward,[37,38] no clear estimate could be given beforehand on this value.
2.2.2 Cloning of (R8)-[A3G2-60]-[I-60] and [I-60]-[A3G2-60]-(R8)
For cloning the repetitive ELP sequences, we chose a method called recursive directional ligation (Figure 2.1A).[39] This technique is based on the scar-free addition of sequences by utilizing type II restriction
Figure 2.1. Overview of recursive directional ligation (RDL) and used ELP sequences. A) RDL extends a given sequence by digestion with either AcuI or BseRI, both type II restriction enzymes, which generate the same overhang, and BglI (step 1 and 2). Combining parts generated in step 1 and 2 (step 3) results in the seamless fusion of both reading frames. Adapted with permission from reference[40]. B)-D) Multiple cloning site
of B) the modified pET-24a(+) vector, C) the vector containing one penta-repeat of [I-n]
and D) [A3G2-n], respectively. Recognition sites of AcuI and BseRI are marked in color, the cutting sites with resulting overhangs are marked with black lines.
A pET-24a(+) vector was modified to include cutting sides for AcuI and BseRI and a C-terminal tyrosine for spectrometric protein detection at 280 nm (Figure 2.1B). A double-stranded DNA oligonucleotide (dsDNA) was designed to encode 5 ELP pentamers compatible with overhangs created by BseRI. After annealing, oligomers were ligated into the modified pET-24a(+) vector and transformed into E. coli XL1-Blue cells (Figure 2.1C, D). Colonies were selected based on the number of inserts and sequenced. In the case of isoleucine, a clone with 15 pentapeptides ([I-15]) and for alanine/glycine a clone with 10 ([A3G2-10)] pentapeptides was successfully selected.Care was taken to ensure full sequencing of the initial inserts (data not shown), since G/C rich sequences often limit the sequencing depth obtained for longer constructs. This was evident in the sequential cloning steps towards the final constructs,[A3G2-60]-[I-60] and [I-60]-[A3G2-60], where successful cloning was confirmed by test- digestion and partial sequencing of the plasmid (Figure 2.2).
Figure 2.2. Visualization of plasmids digested with XbaI and BamHI on a 1.5 % agarose gel. Arrows indicate the DNA length in base pairs as visualized with a marker. Both excised fragments coding for the ELP monoblocks have a length of 966 bp, while the diblocks have a length of 1866 bp and 1893 bp without and with R8, respectively. The digested modified pET24a(+) vector has a length of 5232 bp.
Sequences for octa-arginine were first inserted into the modified pET- 24a(+) plasmid and added to either the N- or C-terminus of ELP diblocks, resulting in R8-[A3G2-60]-[I-60] and [I-60]-[A3G2-60]-R8.
2.2.3 Expression and purification
After sequence design and cloning, a suitable expression host, culture conditions and purification protocols had to be chosen. Bacterial hosts such as E. coli are easily grown in shaken liquid cultures at various conditions.[41] Strains exist that are deficient in recombination factors, RNAses and/or proteases to enhance plasmid, RNA and protein stability. Expression can be either externally induced or auto-induced by co- activation of transcription factors during metabolic shift.[42] E. coli BL21(DE3), BLR(DE3) or BL21Star(DE3) strains were used. BL21(DE3) is a strain deficient in Lon and OmpT proteases.[43] It carries a phage construct that expresses T7 RNA polymerase under the control of a lacUV5 operon.[44] A derivative of BL21(DE3) is BLR(DE3); it is recombinase A deficient[45] and should therefore ensure better stability of repetitive plasmids during culturing. BL21Star(DE3) has reduced levels of RNaseE when compared to BL21(DE3). As plasmids become unstable once transcription is initiated, enhanced RNA stability is expected to increase recombinant protein yield. After optimization, the most reproducible and effective culture conditions were: shaken cultures of 600-1000 mL in baffled 2.5 L flasks with membrane seals, 250-350 rpm at 30-37 °C for 24-30 hours in terrific broth (TB) medium containing 5-8 g/L glycerol, 150 mg/L kanamycin, 0.5 g/L D-glucose and 2 g/L alpha- lactose. This medium is auto-inducing: glucose is the preferred carbon source of E. coli and its use inhibits lactose-related genes and transcription factors. Once glucose is depleted, the metabolic shift to lactose consumption[42] induces T7 RNA polymerase expression, transcription and translation of the ELP coding sequence within the pET24a(+) vector.
Table 2.1 shows the yield of various constructs in the different E. coli strains, as far as tested, under the same expression conditions. For historical reasons, most expressions were done with the BLR(DE3) strain, however more recent results for BL21(DE3) and BL21Star(DE3) indicate the superiority of these strains even for repetitive biopolymers like ELPs. For cell lysis, different methods were compared: enzymatic lysis with hen egg lysozyme, mechanical disruption by sonication and mechanical disruption by cell-homogenizers. While the combination of enzymatic lysis and sonication yielded comparable results to high pressure
homogenization (data now shown), the latter technique was more suitable for larger culture volumes. After cell lysis and DNA precipitation, ELP aggregation was induced by i) changes in temperature or ii) adding salts in solid form or as solution.
Table 2.1. Average expression yields of ELPs in different E.coli strains after purification. Reported values are averages of at least three independent expressions, unless indicated otherwise.